Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September-October 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Principles of method if other than guideline:
Female rats were treated by oral gavage on pregnancy days 6 to 15.
The females were killed on day 20 of pregnancy and a necropsy was performed.
The foetuses were subjected to detailed external, visceral and skeletal examinations.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Piperazine
EC Number:
203-808-3
EC Name:
Piperazine
Cas Number:
110-85-0
Molecular formula:
C4H10N2
IUPAC Name:
piperazine
Specific details on test material used for the study:
Piperazine phosphate (ASCAREX P) was used, CAS 1951-97-9 / EC 217-775-8
Batch no. 1025
100% pure
Expiry date June 1988
1500 g of the test material, a fine white crystalline powder, was received by Toxicol Laboratories on 24th June 1986. The material was stored in the dark and at ambient temperature.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl : CD (SD) BR
Details on test animals or test system and environmental conditions:
One hundred and fifty one female (bodyweight range on arrival 184-221g) rats of the Crl : CD (SD) BR strain were supplied by Charles River (UK) Limited.
Stock males of the same strain were used for mating. For the study, ninety six mated females were selected. The remaining females were discarded undosed.

The animal room was illuminated by fluorescent light with a 24 hour cycle of 12 hours light and 12 hours dark. The room was air-conditioned with equipment set to give 16 air changes per hour and maintain the temperature at 22 +/- 3°C and a relative humidity of 50 +/- 20%.

During the acclimatisation and mating periods the animals were group housed in grid bottomed polypropylene cages suspended above polypropylene trays lined with paper. Mated females were individually housed in solid bottomed polypropylene cages, with wood sawdust provided as bedding.
Tap water, drawn directly from the mains, was provided ad libitum. The animals were fed SQC R and M No. 3 expanded diet (manufactured by Special Diet Services, Essex, U.K.) ad libitum. A certificate of analysis was supplied with each batch of diet. The water authorities supplied certificates of analysis. There were no known contaminants in the food and water that were considered likely to interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The test material was suspended in 1% w/v methyl cellulose (vehicle). Preparation took place daily and separate formulations were prepared for each dose level.
The dose levels were 250, 1000 and 5000 mg/kg bw/day of piperazine phosphate, corresponding to 105, 420, and 2100 mg/kg bw/day piperazine base. \
The females were dosed once daily by the oral route from days 6 to 15 of pregnancy using a rubber catheter and calibrated syringe. A standard dose volume, calculated on an individual bodyWeight basis and adjusted daily, of 10ml/kg bodyweight was given. The control group received the vehicle, 1% w/v methyl cellulose, only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On two occasions, samples were taken from the formulations for each dose level and analysed (by the sponsor) for concentration. The mean assay results for all samples were within 5.0% of the theoretical concentration, demonstrating satisfactory preparation of the dosing formulations.
Details on mating procedure:
After an acclimatisation period of ten days, a male was introduced into each cage of females (1M : 3F). Mating was detected by observation of a sperm positive vaginal smear. The day of observation of sperm was termed day 0 of pregnancy. Mating took place over four days.
Duration of treatment / exposure:
Treatment during pregnancy days 6-15.
Frequency of treatment:
Daily on pregnancy days 6-15.
Duration of test:
the dams were killed on day 20 of pregnancy.
Doses / concentrationsopen allclose all
Dose / conc.:
105 mg/kg bw/day (actual dose received)
Remarks:
calculated as piperazine base
Dose / conc.:
420 mg/kg bw/day (actual dose received)
Remarks:
calculated as piperazine base
Dose / conc.:
2 100 mg/kg bw/day (actual dose received)
Remarks:
calculated as piperazine base
No. of animals per sex per dose:
24 per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Ninety six timed-mated females were allocated to four dose groups on day 0 of pregnancy by a randomisation procedure based on bodyweight.
The dose ranging study (Toxicol report ref. RCT/5/86) demonstrated that the highest dose level selected should elicit minimal maternal toxicity. The lowest dose level selected is a small multiple of the proposed human therapeutic dose and was expected to elicit no maternal toxicity.

Examinations

Maternal examinations:
Clinical observations were recorded daily throughout the study. Body weights were recorded on days 0, 6 to 15 and 20 of pregnancy. Food consumption was measured over the following periods: days 0 to 6, 6 to 11, 11 to 15 and 15 to 20 of pregnancy.
On day 20 of pregnancy all females were killed by C02 asphyxiation and a necropsy was performed.
Maternal tissues showing macroscopic lesions were removed and fixed in buffered 10% formalin.
Ovaries and uterine content:
Abnormalities of major maternal organs.
2. Number of corpora lutea.
3. Number and distribution of implantation sites. The implantations
were classified as early resorptions, late resorptions, dead
foetuses or live foetuses.
Blood sampling:
Not performed.
Fetal examinations:
Live fetal weights, fetal sexes, external abnormalities of fetuses, and visceral and skeletal examinations
Two thirds of the live foetuses from each litter were placed in 70% alcohol. After light fixation, they were skinned, dissected and the viscera were examined. They were then eviscerated and the carcasses processed through potassium hydroxide solution for clearing, alizarin red S for staining the ossified skeleton and aqueous glycerol for preservation and storage. The skeletons were then examined. The bones in each foetus were identified and examined for normality with respect to shape, size and the extent of ossification.
The remaining foetuses were fixed in Bouin's fluid. When fixation was complete the foetuses were sectioned by the Wilson technique and examined for abnormalities.
Structural congenital abnormalities that impair or potentially impair the survival or fitness of the foetus were regarded as major abnormalities. Other defects were classified as minor abnormalities. Commonly observed variations in the degree of ossification from that expected of a day 20 gestation foetus were recorded as variants.
Statistics:
Group mean maternal body weights and food consumption, number of corpora lutea, number of live foetuses, total number of implantation sites and foetal bodyweights (analysed on a litter basis) were subjected to analysis of variance and Student's t test. Maternal bodyweight gain was subjected to Kruskal-Wallis test.
The percentage of foetuses in each litter exhibiting each category of variant, minor abnormality or major abnormality was calculated. ‘The percentage of foetuses in each litter exhibiting one or more miner or one or more major abnormalities for both external/visceral and skeletal abnormalities was calculated. The group mean percentages were calculated from the litter means and the results for control and treated groups compared by Kruskal-Wallis test.
The levels of statistical significance were set at p < 0.05, 0.01 and 0.001.
Indices:
Pre- and post-implantation loss, and sex ratio.
Historical control data:
Toxicol have experience in the use of the Crl : CD (SD) BR strain in reproduction studies and background data on the rate of spontaneous foetal abnormalities have been accumulated.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment related clinical signs were observed at 5000 mg/kg/day only. All females showed excessive salivation immediately before and/or after dosing, towards the end of the treatment period. In addition, two females were observed with wet abdominal fur and/or stained fur around the nose and mouth, and, for one day only, one female was observed to be lethargic and hypothermic.
Mortality:
no mortality observed
Description (incidence):
There were no maternal deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Piperazine phosphate treatment had an adverse effect on maternal body weights at 5000 mg/kg/day. Body weight gain was significantly reduced (p < 0.05, 0.01 or 0.001, Kruskal-Wallis test) in comparison with the controls throughout the dosing period. Reduction in body weight gain was 7% at day 15. After day 15 there was a recovery, although body weights on the day of necropsy were lower than observed for the control group.
Bodyweights at 250 and 1000 mg/kg/day were similar to the controls throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Piperazine phosphate treatment had an adverse effect on maternal food consumption at 5000 mg/kg/day. Consumption was significantly reduced (p < 0.05 or 0.001) throughout the treatment period. Reduction in food consumption was 14% during days 6-11 and 9% during days 11-15. After day 15, food consumption was similar to the controls. There were no adverse effects on food consumption at 250 or 1000 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No females with internal abnormalities were found. One female was observed with an external abnormality, which was considered not to be treatment related: Female 91 (5000 mg/kg/day) showed alopecia on both forelimbs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites were similar to the controls.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post implantation loss was similar to the controls. Pre-implantation loss at 5000 mg/kg/day was less than the controls; however, the difference was not statistically significant and was considered to be fortuitous.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of live foetuses were similar to the controls.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant females was 22, 23, 21 and 22 in groups 1, 2, 3 and 4, respectively.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
420 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity at the next higher dose (clinical signs, reduced body weight gain and food intake)
Remarks on result:
other: piperazine base

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal weights were adversely affected by Piperazine phosphate treatment at 5000 mg/kg/day; weights were significantly lower (7%, p < 0.01) than the controls.
At 250 and 1000 mg/kg/day, fetal weights were similar to the controls.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No effects observed in litter size.
Anogenital distance of all rodent fetuses:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No foetuses with major abnormalities were observed..The overall incidence of minor external abnormalities was not adversely affected by piperazine phosphate treatment; the incidence at 1000 and 5000 mg/kg/day was slightly less than observed in the control group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No foetuses with major abnormalities were observed. The overall incidences of minor skeletal abnormalities were similar for all groups. There were occasional intergroup differences in the incidence of some minor abnormalities and variants of ossification, such as bilobed thoracic centre, but these were not part of a consistent pattern and were considered to be unrelated to treatment. One statistically significant difference was observed: the variation in the incidence of foetuses with hyoid bone not ossified was significant (p < 0.05, Kruskal-Wallis test) when all the groups were compared; however, there were no significant differences when the incidence for each treated group was individually compared with the controls, so this was considered not to be treatment related.
Visceral malformations:
no effects observed
Description (incidence and severity):
No foetuses with major abnormalities were observed. The overall incidence of minor visceral abnormalities was not adversely affected by piperazine phosphate treatment; the incidence at 1000 and 5000 mg/kg/day was slightly less than observed in the control group. The incidences of individual categories of minor abnormalities were within normal ranges for all groups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
420 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: piperazine base
Dose descriptor:
NOAEL
Effect level:
>= 2 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of teratogenicity
Remarks on result:
other: piperazine base

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
2 100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no

Applicant's summary and conclusion

Conclusions:
Pre-implantation loss, post-implantation loss, litter size and fetal sex ratios were not affected by Piperazine phosphate treatment. Evidence of embryotoxicity was limited to the observation of significantly lower fetal weights at 5000 mg/kg/day. There was, however, no evidence of teratogenicity at the dose levels investigated; the incidence of foetal abnormalities was similar for all groups.
Executive summary:

Oral administration of Piperazine phosphate elicited maternal toxicity at 5000 mg/kg/day, observed as a reduction in bodyweight gain and food consumption throughout the treatment period and the occurrence of excessive salivation at the time of dosing. At 250 and 1000 mg/kg/day there were no signs of maternal toxicity.

Evidence of embryotoxicity was limited to the observation of significantly lower fetal weights at 5000 mg/kg/day. This was not accompanied by a retardation of foetal ossification. The effect on fetal weight was considered to be related to the reduced maternal food consumption and not a direct effect of Piperazine phosphate treatment.

Although maternal toxicity and foetal weight retardation were observed, Piperazine phosphate treatment at the specified levels during the period of organogenesis showed no evidence of teratogenicity.

The NOAEL of maternal toxicity was 1000 mg/kg/day piperazine phosphate (420 mg/kg bw piperazine base); the NOAEL of developmental toxicity was also 1000 mg/kg/day piperazine phosphate (420 mg/kg bw piperazine base).

Categories Display